PROJECT SUMMARY A fundamental question in neuroscience is how changes in gene expression are translated into changes in neuronal physiology and ultimately into changes in behavior. The brain's 24-hour timing mechanism, or biological clock, is a neural system that is uniquely suited to the study of the genes-to-behavior problem. A key gap in our knowledge is how the cycling of the neurophysiological rhythms in clock neurons is interconnected with the molecular clockworks to ultimately drive behavior. We conceptualize the clockworks in neurons as being composed of 3 interconnected layers or levels ? genes, membrane and messengers. Here, we hypothesize that the neurophysiological and TTFL levels of the clock are coordinated through mutual reinforcement of the membrane and gene level rhythms, as mediated by the messenger Ca++, and CREB. We will use transgenic circadian reporter mice combined with optogenetics to address the following questions - How does manipulation of SCN neuron spike frequency modify the phase and period of the TTFL? We have successfully instituted both stimulatory and inhibitory optogenetic control of SCN neuron spike frequency, and used this approach to modify the phase and period of the SCN TTFL in vitro. Here we will determine (i) the mechanisms by which altering SCN spike frequency resets the phase of the SCN clock by examining membrane Ca++ fluxes, CamKII or MAPK activation, CREB/CRE induction and (ii) whether the ongoing membrane spike frequency rhythm is necessary to maintain molecular TTFL rhythmicity. How does altering SCN neuron spike frequency affect organization of the SCN? We have found that optogenetic spike- frequency-mediated clock resetting requires intercellular communication via SCN neuron action potentials and network communication through VIP. Here we will determine the mechanisms underlying SCN network effects of optogenetic stimulation and whether additional network communication through GABA and AVP signaling may be critical. In addition, (ii) we will define how spike frequency induced resetting may reconfigure the SCN network to alter free-running period as well as phase. Can manipulation of in vivo SCN spike frequency per se encode circadian behavior? We have used optogenetic stimulation of the SCN in vivo to reorganize circadian behavior and will use this approach to test for rescue of behavioral rhythms in arrhythmic backgrounds and whether optogenetic manipulation of the daily SCN spike frequency profile can mimic photoperiod encoding. Successful completion of these aims will provide novel insight into mechanisms by which SCN neurons generate and sustain coherent 24-hour rhythms that drive behaviors. As the clock genes are widely expressed in the brain and contribute to neuronal excitability in learning and memory and addiction, this work will also point to general mechanisms of genetic control of neuronal function and behavior. In addition, successful definition of how SCN spike frequency may encode circadian behavior and photoperiod would open the way for manipulation of SCN activity as a future therapeutic approach for circadian disorders.